The capacity for oxidative metabolism is a fundamental property of the differentiated state that varies widely among animal cells and tissues. Differences in respiratory function in both normal cells and in a variety of cellular pathologies are reflected in the number, size, and morphology of mitochondria as well as in the expression of respiratory gene products. A fundamental issue is how the biosynthetic activities of nuclear and mitochondrial genetic systems are coordinated to meet cellular energy demands Much is known about the organization and expression of the mammalian mitochondrial genome However, little is known about the nuclear genes which encode the majority of respiratory subunits and all of the gene products required for mitochondrial transcription and replication. The cytochrome c genes have been developed as a model for exploring the nuclear control of respiratory gene expression. These studies have led to the discovery of nuclear respiratory factors (NRFs). The evidence strongly supports a role for these factors in the expression of many recently isolated genes encoding subunits of the oxidative complexes and a component of the mitochondrial DNA replication machinery. The long term objective of the current proposal is to define the molecular interactions and physiological functions of NRFs and related activator proteins. The specific aims are: 1) To complete an extensive mutational and nuclear factor binding analysis of the cytochrome c promoter. Emphasis will be placed upon a potent upstream transcriptional enhancer designated region I/NRF-1 and the identification of regulatory factor(s) with which it interacts. 2) To identify cis and trans-regulatory elements mediating expression of the cytochrome c oxidase subunit IV (COXIV) gene. The emphasis here will be upon NRF-2, a factor which recognizes and directs transcription through related sequences in the COXIV and the ATP synthase beta-subunit (betaATPS) genes. 3) To purify NRF-1 and NRF-2 and to isolate the cDNA clones encoding these factors. The transcriptional and DNA binding specificities of the cloned NRF factors will be established and compared to their nuclear counterparts. 4) To utilize cellular systems amenable to molecular analysis to define physiological pathways of respiratory gene expression and the roles of NRF-1 and NRF-2. Responses to signals such as thyroid hormones and growth factors will be assigned to specific cytochrome c and COXIV regulatory elements. The analytical probes developed through the molecular cloning of NRF-1 and NRF-2 will be used to investigate the relationship between NRF regulation (both transcriptional and post-transcriptional) and the genes they control.